Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.     

Molecular approaches to the identification and treatment monitoring of periodontal pathogens

Two different PCR-based molecular approaches, a commercial kit for detection of A. actinomycetemcomitans, P. gingivalis, P. intermedia, B. forsythus and T. denticola (Amplimedical "Paradonthosis") and a home-made multiplex PCR for A. actinomycetemcomitans, P. gingivalis and B. forsythus were compared for monitoring the efficacy of different dental treatments on localized persistent periodontal pockets. 44 sites were randomized in two treatment groups: mechanical treatment (22 control sites) and in conjunction with the application of tetracycline fibres (22 experimental sites). 40/44 sites were found positive with both tests for A. actinomycetemcomitans, P. gingivalis and B. forsythus pretheraphy. P. intermedia was detected alone in only three sites during the follow-up, while T. denticola. was always associated with the other pathogens. 20 sites were positive in conventional cultures for one to three of the pathogens. PCR-based approaches provided a sensitive and reliable method for identification and monitoring treatment of periodontal pathogens.     

甘草提取物对四种牙周常见致病菌的抑菌作用

目的体外评价甘草提取物对牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌四种牙周常见致病菌的抑制效果。方法以牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌四种牙周常见致病菌作为供试菌,采用液体稀释法,考察甘草提取物对这四种细菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC);并采用不同浓度的甘草提取物溶液,绘制甘草提取物对四种牙周致病菌的时间-杀菌曲线。结果甘草提取物对牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌的MIC值分别为1.50、1.50、0.75和1.50mg/mL,MBC值分别为6、3、3和3mg/mL。当甘草提取物达到对四种细菌的MBC值时,对于牙龈卟啉单胞菌、中间普氏菌、伴放线放线杆菌可在2h后可达到杀菌效果,对于具核梭杆菌可在4h后达到杀菌效果。结论甘草提取物对以上四种牙周常见致病菌具有良好的抑菌及杀菌作用。     

Salivary detection of periodontopathic bacteria in periodontally healthy children

BACKGROUND: Salivary occurrence of periodontopathic bacteria is of interest especially in children as a risk indicator for the transmission, development and control of periodontal disease. We assessed the prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Prevotella nigrescens and Treponema denticola as microbial complexes in the saliva of children with mixed dentition and healthy gingiva. MATERIALS AND METHODS: Paraffin-stimulated saliva samples were collected from 41 children (22 boys and 19 girls), aged 6-13 years old. Gingival health was determined during the initial screening exam. The test bacteria were identified using a 16S rRNA-based PCR analysis. RESULTS: P. nigrescens was the most frequent species (80%), followed by T. denticola (32%), A. actinomycetemcomitans (24%) and P. gingivalis (12%). P. intermedia and T. forsythia were not detected. P. nigrescens was also common species in combinations. Paired and triple bacterial combinations were found in 24% and 20% of all children, respectively. There was no positive association between bacterial combinations in colonization and subject's gender (P>0.05, Fisher exact test). CONCLUSION: The salivary presence of P. nigrescens, T. denticola, A. actinomycetemcomitans and P. gingivalis but not P. intermedia and T. forsythia can occur in childhood without clinical signs of gingival disease. Thus, the possible risk of bacterial transmissions through saliva and, the need to screen for periodontal pathogens should be considered before mixed dentition.     

Molecular analysis of the subgingival microbiota in health and disease

This investigation provides molecular analyses of the periodontal microbiota in health and disease. Subgingival samples from 47 volunteers with healthy gingivae or clinically diagnosed chronic periodontitis were characterized by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the V2-V3 region of the eubacterial 16S rRNA gene. A hierarchical dendrogram was constructed from band patterns. All unique PCR amplicons (DGGE bands) were sequenced for identity. Samples were also analyzed for the presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis by multiplex PCR. Associations of patient age, gender, and smoking status together with the presence of each unique band and putative periodontal pathogens with disease were assessed by logistic regression. Periodontal pockets were colonized by complex eubacterial communities (10 to 40 distinct DGGE bands) with substantial individual variation in the community profile. Species diversity in health and disease was determined by the Shannon-Weaver index of diversity and compared by the Mann-Whitney U test. Sequence analyses of DGGE amplicons indicated the occurrence of many nontypical oral species and eubacteria previously associated with this environment. With the exception of T. forsythensis, the putative pathogens were not detected by DGGE. Multiplex PCR, however, detected T. forsythensis, A. actinomycetemcomitans, and P. gingivalis in 9% 16%, and 29% of the patients with disease, respectively. The presence of A. actinomycetemcomitans was significantly associated with disease (P < 0.01). Statistical analyses indicated that the presence of Treponema socranskii and Pseudomonas sp. was a significant predictor of disease (P < 0.05) and that there was no significant difference (P > 0.05) in terms of eubacterial species diversity between health and disease.     

Periodontitis in established rheumatoid arthritis patients: a cross-sectional clinical,microbiological and serological study

Introduction

The association between rheumatoid arthritis (RA) and periodontitis is suggested to be linked to the periodontal pathogen Porphyromonas gingivalis. Colonization of P. gingivalis in the oral cavity of RA patients has been scarcely considered. To further explore whether the association between periodontitis and RA is dependent on P. gingivalis, we compared host immune responses in RA patients with and without periodontitis in relation to presence of cultivable P. gingivalis in subgingival plaque.

Methods

In 95 RA patients, the periodontal condition was examined using the Dutch Periodontal Screening Index for treatment needs. Subgingival plaque samples were tested for presence of P. gingivalis by anaerobic culture technique. IgA, IgG and IgM antibody titers to P. gingivalis were measured by ELISA. Serum and subgingival plaque measures were compared to a matched control group of non-RA subjects.

Results

A higher prevalence of severe periodontitis was observed in RA patients in comparison to matched non-RA controls (27% versus 12%, p < 0.001). RA patients with severe periodontitis had higher DAS28 scores than RA patients with no or moderate periodontitis (p < 0.001), while no differences were seen in IgM-RF or ACPA reactivity. Furthermore, RA patients with severe periodontitis had higher IgG- and IgM-anti P. gingivalis titers than non-RA controls with severe periodontitis (p < 0.01 resp. p < 0.05), although subgingival occurrence of P. gingivalis was not different.

Conclusions

Severity of periodontitis is related to severity of RA. RA patients with severe periodontitis have a more robust antibody response against P. gingivalis than non-RA controls, but not all RA patients have cultivable P. gingivalis.     

Periodontal status in an Italian young adult population. Prevalence and relationship with periodontopathic bacteria

To determine the prevalence of periodontitis in an Italian young adult population and the relationship with Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in subgingival plaque. A full-mouth periodontal and oral examination was performed in 70 subjects. Dental and behaviour habits were assessed with a standardised questionnaire. Subgingival plaque samples were collected from the deepest pocket of the first molars in each quadrant with a sterile curette. A. actinomycetemcomitans, P. gingivalis and P. intermedia were detected using a multiplex polymerase chain reaction. At subject level, the prevalence of bleeding on probing, calculus, normal pocket depth (PD), PD > 5mm and bacterial positivity were 44.8%, 43.3%, 22.9%, 11.4% and 95.7%, respectively. At quadrant level bacterial prevalence was 79.4%; P. intermedia was the most common bacteria (79.0%); A. actinomycetemcomitans had a prevalence of 40.8%. A significant linear trend across categories of gingival conditions (healthy, bleeding on probing, calculus presence) was detected for P. intermedia (p = 0.0038) and A. actinomycetemcomitans (p = 0.00005) proportions. No significant association was observed between pathogenic bacteria and PD, nor with behavioural attitudes. Gingival conditions are found to be a good predictors (VPP = 85%) for periodontopathic bacteria. For the Italian population, as no data are present, prospective longitudinal studies are needed to examine the relationship between PD and bacteria presence with periodontal disease onset and/or progression.     

Quantification of Subgingival Bacterial Pathogens at Different Stages of Periodontal Diseases

Anaerobic gram-negative oral bacteria such as Treponema denticola, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Campylobacter rectus, and Fusobacterium nucleatum are closely associated with periodontal diseases. We measured the relative population (bacterial levels) of these oral pathogens in subgingival tissues of patients at different stages of Korean chronic periodontal diseases. We divided the individuals into those with chronic gingivitis (G), moderate periodontitis (P1), severe periodontitis (P2), and normal individuals (N) (n?=?20 for each group) and subgingival tissue samples were collected. We used real-time PCR with TaqMan probes to evaluate the change of periodontal pathogens among different stages of periodontitis. Bacterial levels of A. actinomycetemcomitans and C. rectus are significantly increased in individuals with chronic gingivitis and moderate periodontitis, but unchanged in severe periodontitis patients. These results suggest that analyzing certain bacterial levels among total oral pathogens may facilitate understanding of the role of periodontal bacteria in the early stages of periodontitis.     

Use of molecular genetic systems for diagnostics of inflammatory diseases of oral mucosa and periodontium

Structure of bacterial microflora of periodontal pocket using classical cultural and molecular genetic methods (PCR and RT-PCR assays) was studied in patients with chronic generalized severe or intermediate periodontitis in acute phase and in healthy subjects. Structure of viral and fungal microflora using PCR or ELISA assays was studied also. Markers of Gram-negative bacteria (B. forsithus, T. denticola, A. actinomicetemcommitans, P. intermedia and P. gingivalis) were detected with different rates. Rate of detection of pigment-forming P. intermedia and P. gingivalis was significantly higher when PCR assay was used compared with traditional bacteriologic method. Detection rate of Candida albicans DNA in tooth-gingival fluid was virtually not different from one when cultural method was used. PCR assay is recommended as laboratory method for detection of DNA of microorganisms associated with inflammatory diseases of oral mucosa and periodontium.     

口腔卫生预防措施对固定正畸患者牙周菌群和牙周临床指数的影响

目的 采用牙周洁治和强化的口腔健康教育方法,通过检测牙周菌群和牙周临床指数的动态变化,探讨口腔卫生预防措施对固定正畸患者牙周菌群的影响。方法 选择正畸初诊患者20例,平均分为试验组（T组）和对照组（C组）。于正畸治疗开始前检查右上颌第一磨牙16和右下颌中切牙41牙周状况,并采集其近中颊轴嵴处龈缘菌斑做细菌分离培养。T组于治疗前进行全口洁治,每月复诊加力时均给予口腔卫生检查,强调口腔卫生的重要性;C组仅在初诊时进行口腔卫生指导,其余不作处理。2组患者分别于矫治器安装后1、3和6个月进行临床及细菌学检查。结果 随观察时间的延长,T组颊侧菌斑指数和牙龈指数在第1、3、6个月时较基线降低;C组颊侧菌斑指数第6个月时较基线降低,舌侧探诊深度则升高（P＜0.05）。细菌检出率和检出量的变化在T组可见韦荣球菌属降低而弯曲杆菌属和Gn产黑色素厌氧杆菌（BPAR）升高,C组消化链球菌属和BPAR升高（P＜0.05）;BPAR在第3个月、消化链球菌属在第6个月时T组检出率低于C组（P＜0.05）,而细菌检出量和牙周临床指数在2组间没有观测到处理因素的作用（P＞0.05）。结论 正畸前即存在牙龈炎的患者,建议进行预防性牙周洁治;牙周洁治必须和口腔卫生教育、正确的日常菌斑控制措施结合进行。     

成人牙周炎龈下套氧菌分离鉴定及药敏试验结果分析

分离并鉴定了329例成人牙周炎龈下优势厌氧菌群,并对不同病程中的菌群变迁、厌氧菌的药物敏感性进行了分析.成人牙周炎龈下标本中厌氧菌阳性检出率为97.9%,其中以牙龈紫质单胞菌检出率最高(38.5%),具核梭杆菌次之(18.9%).随着牙周病变程度的加重,牙龈紫质单胞菌、具核梭杆菌、产黑色素普氏菌、星群厌氧链球菌、厌氧消化链球菌的检出率增高(P＜0.05),小韦荣球菌的检出率下降(P＜0.01),表明前5种厌氧菌在AP发病过程中有重要作用,小韦荣球菌与之无关.替硝唑、甲硝唑和克林霉素对438株革兰氏阴性厌氧菌的MIC90分别为1～8,2～8和4～16 mg/L,对278株革兰氏阳性厌氧菌的MIC90分别为16～32,16～64和4～16 mg/L,表明替硝唑和甲硝唑体外抗革兰氏阴性厌氧菌效果优于克林霉素,抗革兰氏阳性厌氧菌作用不如克林霉素.     

Use of DNA probes in the diagnosis and treatment of periodontitis --a case series

Aggressive periodontitis is characterized by rapid attachment and bone loss with no underlying systemic disease and is associated with specific bacteria like Actinobacillus actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg). In this case series 25 patients were diagnosed with aggressive periodontitis by the aid of DNA probes for Aa and Pg and other periodontal pathogens. The use of DNA probes for the detection of periodontal pathogens may aid in the diagnosis and treatment of aggressive periodontitis. Clinical experience suggests that lowering periodontal pathogens to undetectable levels could improve the long-term stability of periodontal health.     

Genetic variation of a fimbrial protein from Porphyromonas gingivalis and its distribution in patients with periodontal diseases

Pg-II fim from various strains of Porphyromonas gingivalis was classified on the basis of each nucleotide sequence, while the distribution of Pg-II fim types in 141 subgingival plaque samples was analyzed using PCR assays. Pg-II fim was divided into two types as follows: strains OMZ409, HG405, 381, ATCC 33277 and BH18/10 (type 1) and strains OMZ314 and HW24D-1 (type 2). The presence of P. gingivalis was demonstrated in 2.8% of healthy subjects and 56.1% of patients with periodontal diseases, and Pg-II fim was detected in 91.8% of the P. gingivalis-positive subjects. We also analyzed the distribution of the Pg-II fim types among Pg-II fim-positive patients, with the following results: type 1 (38.2%), type 2 (56.4%) and types 1 and 2 (5.4%). These findings strongly suggest that P. gingivalis organisms possessing Pg-II fim type 2 was principally detected in patients with periodontal diseases.     

固定矫治器戴入前后牙周可疑病原菌和牙周临床指数的变化

目的:固定矫治器戴入前后的不同时间进行龈缘菌斑的微生物学检查和牙周状况的临床检查,以探讨固定矫治器戴入前后牙周可疑病原菌和牙周状况的动态变化过程.方法:选择18名固定正畸患者,于矫治器戴入前和戴入后1、3、6月分别检查16、41牙位的菌斑指数、牙龈指数、探诊深度、探诊出血,并在近中颊侧颊轴角处采集龈缘菌斑样本,采用细菌培养鉴定方法测定牙周可疑病原菌的检出量(CFU/g)和检出率.结果:与基线相比,观察期内龈缘菌斑的G￣产黑色素厌氧杆菌检出量和检出率在两个牙位均升高(P＜0.05),41的优杆菌、弯曲杆菌、拟杆菌、普氏菌亦有升高(P＜0.05).临床指标中16的牙龈指数(颊侧)和探诊深度(颊、舌侧)升高;41的菌斑指数(舌侧)降低(P＜0.05).结论:固定矫治器戴入后虽然可通过严格的口腔卫生指导有效控制牙面菌斑,但仍可引起牙周可疑病原菌增加.     

8-oxo-7,8-dihydroguanine is removed by a nucleotide excision repair-like mechanism in Porphyromonas gingivalis W83

A consequence of oxidative stress is DNA damage. The survival of Porphyromonas gingivalis in the inflammatory microenvironment of the periodontal pocket requires an ability to overcome oxidative stress caused by reactive oxygen species (ROS). 8-oxo-7,8-dihydroguanine (8-oxoG) is typical of oxidative damage induced by ROS. There is no information on the presence of 8-oxoG in P. gingivalis under oxidative stress conditions or on a putative mechanism for its repair. High-pressure liquid chromatography with electrochemical detection analysis of chromosomal DNA revealed higher levels of 8-oxoG in P. gingivalis FLL92, a nonpigmented isogenic mutant, than in the wild-type strain. 8-oxoG repair activity was also increased in cell extracts from P. gingivalis FLL92 compared to those from the parent strain. Enzymatic removal of 8-oxoG was catalyzed by a nucleotide excision repair (NER)-like mechanism rather than the base excision repair (BER) observed in Escherichia coli. In addition, in comparison with other anaerobic periodontal pathogens, the removal of 8-oxoG was unique to P. gingivalis. Taken together, the increased 8-oxoG levels in P. gingivalis FLL92 could further support a role for the hemin layer as a unique mechanism in oxidative stress resistance in this organism. In addition, this is the first observation of an NER-like mechanism as the major mechanism for removal of 8-oxoG in P. gingivalis.     

Occurrence of Porphyromonas gingivalis with Prevotella intermedia in periodontal samples

Abstract To further examine the previously suggested inverse relationship between Porphyromonas gingivalis and Prevotella intermedia in periodontal disease, 1016 samples taken from single or multiple (pooled) subgingival sites were cultured anaerobically and examined for the simultaneous occurrence of the microorganisms. P. gingivalis was isolated from 297 (29%) and Pr. intermedia from 501 (49%) samples. P. gingivalis was found as frequently with (14%) as without (15%) Pr. intermedia . The type of sampling had no effect on the occurrence of P. gingivalis with Pr. intermedia . However, female subjects harboured them in combination more frequently than male subjects. The mean proportions of P. gingivalis in the cultivable flora appeared to be lower when found with than without Pr. intermedia . Whether the detection of the combination, or P. gingivalis alone, has clinical relevance needs further clarification.     

液相BANA试验检测口腔关键厌氧菌方法的建立

目的建立液相苯甲酰精氨酸萘酰胺(简写BANA)水解试验快速检测牙周病关键厌氧菌的新技术。方法利用牙周病相关关键微生物可产生胰蛋白酶样酶使人工合成的酶底物———BANA水解的原理检测这些重要口腔厌氧菌。结果BANA试验可检测出8.0×105以上的牙龈卟啉菌,特异性良好、不受标本采集中血污染的影响;71.88%活动期牙周炎龈下菌斑BANA试验阳性,4%的健康人龈下菌斑BANA试验阳性。结论BANA试验可望成为牙周病诊断新的辅助指标。     

Detection rates of periodontal bacteria and herpesviruses in different forms of periodontal disease

The aim was to investigate the detection rates of periodontal bacteria (Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans) and herpesviruses (herpes simplex virus-1 [HSV-1], cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) in different forms and severity of periodontal disease, and to compare them with those in periodontally healthy subjects. One hundred and twenty-nine patients participated in the study: 39 diagnosed with periodontal abscess (PA), 33 with necrotizing ulcerative periodontitis (NUP), 27 with chronic periodontitis (CP), and 30 participants with healthy periodontal tissue represented a healthy control group. All patients with periodontal disease (PA, NUP, and CP) were also divided into two groups according to the severity of their disease: moderate and severe periodontitis. The subgingival samples were collected from the periodontitis active sites and the detection of microorganisms was performed by end-point polymerase chain reaction analyses. The results revealed significantly higher detection rates of P. gingivalis, T. forsythia, and P. intermedia in all three groups of patients with periodontitis than in healthy participants. The highest detection rate of A. actinomycetemcomitans was noticed in CP, which was significantly higher than that in PA, NUP, and healthy control. The occurrence of EBV was significantly higher in NUP than in CP and healthy participants. CMV was detected significantly more frequently in PA and NUP than in CP and healthy participants. Comparisons among healthy participants and patients with moderate and severe periodontitis showed significantly higher detection rates of EBV and CMV in patients with severe forms of periodontitis than in healthy participants and those with moderate periodontitis.